Regulated turnover and processing of cellular RNAs is critical in the control of gene expression during cell growth and differenciation. Ribonucleases III form a family of highly- specific double-stranded RNA (dsRNA) endonucleases which control a wide variety of cellular RNA processing pathways. Prokaryotic and eukaryotic RNases III have a conserved important function in the processing of the precursor of ribosomal RNA (rRNA). Eukaryotic RNases III also play critical roles in the processing of a large number of precursors of small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs), which are essential to mRNA splicing, and rRNA processing and metabolism, respectively. Our long term goal is to explore the multiple functions of RNases III in the control of eukaryotic gene expression. The S. cerevisiae RNase III homolog Rnt1p will be studied as a model enzyme to investigate the function of double-stranded RNA cleavage in the control of eukaryotic gene expression, and to decipher the biochemical mechanisms of specific cleavage by dsRNAses. Within this framework, our specific aims are: 1. To exhaustively identify natural RNA targets of yeast RNase III. Genetic results indicate that eukaryotic RNases III specifically controls the level of several specific mRNAs, in addition to their roles in rRNA, snRNA and snoRNA processing. Genomic approaches will be developed to exhaustively identify mRNA substrates of Rnt1p. An exhaustive list of RNA substrates will provide the phylogenetic information regarding conserved structural elements of the RNA substrates, and will shed light on the gene expression pathways controlled by this enzyme. 2. To understand the mechanisms of specific RNA recognition by yeast RNase III. Finding the biochemical determinants of RNA recognition by Rnt1p is an important issue to understand the specificity of RNA targeting by dsRNA endonucleases. Using a combination of biochemical, structural and genetic studies, RNA and protein chemical groups which are responsible for the specificity of RNA recognition by eukaryotic RNase III will be identified. 3. To unravel the integration of yeast RNase III activity in the cell metabolism. Several yeast proteins have been identified which interact with Rnt1p and are expected to influence Rnt1p activity in vivo. The influence of these proteins on Rnt1p activity and subcellular localization will be tested. In particular, provocative hypotheses suggesting that Rnt1p activity is modulated by N- terminal acetylation and that Rnt1p is involved in the sexual process will be tested.